Assessment of cyto-protective, antiproliferative and antioxidant potential of a medicinal plant Jatropha podagrica

Industrial Crops and Products 44 (2013) 111–118

Contents lists available at SciVerse ScienceDirect

Industrial Crops and Products journal homepage: www.elsevier.com/locate/indcrop

Assessment of cyto-protective, antiproliferative and antioxidant potential of a medicinal plant Jatropha podagrica Wafa Ghali a , David Vaudry b , Thierry Jouenne c , Mohamed Néjib Marzouki a,∗ a b c

LIP-MB, National Institute of Applied Sciences and Technology, Centre urbain nord de Tunis, B.P. 676 Cedex Tunis – 1080, University of Carthage, Tunisia U413, Cellular and Molecular Neuroendocrinology Laboratory, (IFRMP 23), Université de Rouen, Mont Saint Aignan, France UMR CNRS 6270, Plate-Forme Protéomique IBiSA (IFRMP 23), FR 3038 CNRS, Université de Rouen, Mont Saint Aignan, France

a r t i c l e

i n f o

Article history: Received 24 May 2012 Received in revised form 27 September 2012 Accepted 18 October 2012 Keywords: Jatropha podagrica Antioxidant activity DNA damage Lipid peroxidation Anti-proliferative Cytotoxicity

a b s t r a c t The present study is aimed to evaluate antiproliferative, antioxidant activity and in vitro protective effect of hydro-alcoholic extract of Jatropha podagrica. Total phenolics and flavonoids content was higher in seeds than leaves extract. The anti-proliferative effect on two tumoral cell lines A549 and PC12 was assessed by MTT assay. The cytotoxicity of the studied extract was also evaluated on young cerebellar granule cells. The investigation of the action mode of J. podagrica extract in protection of DNA damages, protein carbonylation and lipid peroxidation caused by hydroxyl radicals generated in Fenton’s reaction exhibits an effective protection at low concentrations. In the presence of 100 ␮M of copper sulfate, Jatropha hydro-alcoholic extract showed no significant enhancing in the hydroxyl radicals generation and DNA fragmentation in vitro. These results suggest that extract of J. podagrica still acts as antioxidant even in the presence of free metal ions, but with less efficiency. The J. podagrica extract which showed significant antitumor activity against the A549 and PC12 cells deserves further research into the chemoprevention and treatment of cancer. © 2012 Elsevier B.V. All rights reserved.

1. Introduction In recent years, the studies on “oxidative stress” and its adverse effects on human health have become a subject of considerable interest. It is a well-documented fact that exposure of organisms to exogenous and endogenous factors generates a wide range of reactive oxygen species (ROS), resulting in homeostatic imbalance (Halliwell, 1994; Sies, 1997; Halliwell and Gutteridge, 1999). ROS can induce alterations and loss of structural/functional architecture in the cell, leading directly to cytotoxicity and/or indirectly to genotoxicity (Girotti, 1994; Esterbauer, 1996; Sies, 1997; Halliwell and Gutteridge, 1999). Therefore, the factors that shift the physiological process, in the homeostatic balance are of great interest (Sies, 1997). Antioxidant principles from plant resources are multifaceted in their effects and provide enormous scope in correcting the imbalance. Plants (fruits, vegetables, medicinal herbs) contain a wide variety of free radical-scavenging molecules, such as phenolic compounds, nitrogen compounds, vitamins, terpenoids, and some other endogenous metabolites, that are rich in antioxidant activity (Zheng and Wang, 2001; Cai et al., 2003). Biomolecules in fruits

∗ Corresponding author. Tel.: +216 98 500 566; fax: +216 71 704 329. E-mail addresses: [email protected], [email protected] (M.N. Marzouki). 0926-6690/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.indcrop.2012.10.020

and vegetables have attracted a great deal of attention mainly directed to their antimicrobial, anti-proliferative and protecting activities (Fattouch et al., 2008, 2010; García-Pérez et al., 2010). The demand for natural antioxidant phytochemicals of plant origin has increased as they are viewed as promising therapeutic agents for free radical pathologies (Kitts et al., 2000). Jatropha podagrica is a multipurpose shrub in the family Euphorbiaceae commonly found in Africa, Asia and Latin America. Jatropha species are used in traditional medicine for various diseases such as skin infections, sexually transmitted diseases like gonorrhea, jaundice and fever (Burkill, 1994). Few pharmacological activities (antibacterial and anti-insect) have been reported for this plant (Aiyelaagbe et al., 1998). The aim of this study was to screen antioxidant activity of J. podagrica extracts and to investigate the possible protective properties, anti-proliferative effect and the cytotoxicity of different concentrations of Jatropha extract in PC12 and A549 cells. The effect of studied extract was also investigated against induced lipid peroxidation, proteins carbonylation and DNA damages in isolated brain cells. To the best of our knowledge, this is the first study that has investigated proprieties of this medicinal plant. Determining the cytotoxic and anti-proliferative effects of the antioxidants of J. podagrica in a neuronal model is important not only to establish its safety, but also to assess possible hazards when combined with other chemical agents such as the chemotherapy drugs used in cancer therapy.

112

W. Ghali et al. / Industrial Crops and Products 44 (2013) 111–118

2. Materials and methods 2.1. Chemicals Catechin, sodium nitrite (NaNO2 ), aluminum chloride (AlCl3 ), sodium hydroxide (NaOH), gallic acid, Folin–Ciocalteu reagent, sodium carbonate (Na2 CO3 ), trolox, 1,1-diphenyl-2picrylhydrarazyl (DPPH), ferrous sulfate (FeSO4 ·7H2 O), potassium ferrocyanide [K3 Fe(CN)6 ], trichloroacetic acid (TCA), ferric chloride (FeCl3 ), ethylenediamine-tetraacetic acid (EDTA), ferrous chloride (FeCl2 ), ferrozine, riboflavine, nitro blue tetrazolium (NBT), butylated hydroxytoluene (BHT), l-ascorbic acid, methanol, hexane, ethyl acetate, ethanol, dimethyl sulfoxide (DMSO); were all purchased from Sigma Aldrich (St. Louis, MO). 2.2. Plant materials and preparation of extracts The fresh leaves and seeds of J. podagrica were collected, and washed with water to remove all debris. The leaves were dried for 24 h at 70 ◦ C. The dried leaves and seeds were then ground in a mortar with a pestle under liquid nitrogen. The powdered material was, first, defatted with 100 ml of hexane and then extracted using solvent methanol and distilled water in the ratio (80:20) during 48 h. After extracting all coloring matter, the solvent was removed by evaporation to dryness under reduced pressure at room temperature by a Rotavapor; which resulted in a solid mass of each extract. 2.3. Determination of total phenolic compounds Total phenolics were determined using a modified colorimetric Folin–Ciocalteu method (Wolfe et al., 2003) with gallic acid as standard. Briefly, extract (1 mg) and 0.5 ml of water were mixed in a test tube. Folin–Ciocalteu reagent (0.125 ml) was added and the solution was allowed to react for 6 min at room temperature followed by the addition of 1.25 ml of 7.5% Na2 CO3 solution and 2.5 ml of distilled water. After 60 min of incubation at room temperature in the dark, absorbance of the mixture was measured at 760 nm. The analysis was performed in triplicate and total phenolic content was expressed as gallic acid equivalents (GAE) in milligrams per gram sample (dry matter). 2.4. Determination of flavonoids content The flavonoids content was performed according to the colorimetric assay of Dewanto et al. (2002). Test samples (50 ␮l) were mixed with 30 ␮l of 5% sodium nitrite solution for 60 min at ambient temperature, before addition of 60 ␮l of 10% of aluminum chloride (AlCl3 ) freshly prepared. After 5 min at room temperature, 200 ␮l of 1 M NaOH solution was added to the mixture. The volume of the mixture was made up to 1 ml with distilled water. The mixture was thoroughly vortexed and the absorbance was measured at 510 nm. Flavonoids content was expressed as mg of catechin equivalent per gram of dry weight. 2.5. Free radical scavenging capacity (DPPH scavenging assay) Scavenging activity on DPPH free radicals by the examined extract was evaluated by the method reported by Gyamfi et al. (1999). Briefly, 50 ␮l of the hydro-alcoholic extract containing various amounts of powdered extract (1–300 ␮g/ml methanol, respectively in each reaction) were mixed with 1 ml of 0.1 mM DPPH (2,2-diphenylhydrazil)-methanol solution and 450 ␮l of 50 mM Tris–HCl buffer (pH 7.4). After 30 min incubation period at room temperature in the dark, the absorbance of the resulting solution and blank (with same reagents except the samples) was

recorded at 517 nm against ascorbic acid as positive control. Three replicates of samples were recorded. The percentage of inhibition of DPPH free radicals was calculated in the following way:



% Inhibition = 100 ×

Ablank − Asample



Ablank

with Asample is the absorbance of the compound and Ablank the absorbance of the control solution. The IC50 value, which represented the concentration of the tested extracts that caused 50% reduction of the initial DPPH concentration, was calculated from a non linear regression curve of log concentration of tested extract (␮g/ml) against the mean percentage of the radical scavenging activity. 2.6. Reducing power The reducing power of the extracted samples was determined according to the method of Shyamala et al. (2004). Different amounts of extracts/standard (100–400 ␮g) in 1 ml distilled water were mixed with phosphate buffer (2.5 ml, 0.2 M, pH 6.6) and potassium ferricyanid [K3 Fe(CN)6 ] (2.5 ml, 1%). The mixture was heated at 50 ◦ C for 20 min then 2.5 ml of tricholoroacetic acid (TCA) (10%) was added and the whole solution was centrifuged (3000 rpm, 10 min). The upper layer of the solution (2.5 ml) was mixed with distilled water (2.5 ml) and FeCl3 (0.5 ml, 0.1%). Absorbance was measured at 700 nm. Increased absorbance of the mixture indicated an increase in the reducing power. Ascorbic acid was used as standard and all experiments were performed in triplicate. 2.7. Superoxide anions scavenging activity The capacity of scavenging superoxide anions by the extracts was assessed using the method of Beauchamp and Fridovich (1971), with slight modifications. In brief, different amounts of extract/standard were dissolved in phosphate buffer (50 mM, pH 7.6) to have various concentrations (5–400 ␮g/ml). The extract solution was mixed with riboflavin (20 ␮g), EDTA (12 mM) and nitro blue tetrazolium (NBT) (333 mg/ml). The final volume was adjusted to 1.5 ml with phosphate buffer (50 mM, pH 7.6). The samples were illuminated between 90 and 150 s then the absorbance was directly measured at 590 nm against a blank (the blank contains all the chemicals except the extract and was kept in the dark until reading the absorbance). The scavenging/inhibition capacity of superoxide anions by the extract was estimated according to the following formula: % Inhibition =

A − A  0 1 A1

× 100

A0 : absorbance of the control solution and A1 : absorbance of the sample. 2.8. Lipid peroxidation inhibition (ex vivo assay) In order to quantify the concentration of oxidized lipids caused by ROS generation in the brain homogenate, the amount of thiobarbituric acid-reactive substances (TBARS) was determined using the method of Ohkawa et al. (1979) with slight modifications. Cellular homogenate was prepared using a freshly dissected sheep brain. The brain was homogenized with polytron in ice-cold Tris–HCl buffer (20 mM, pH 7.4); the produced 1:2 (w/v) homogenate tissue was centrifuged at 3000 × g for 10 min. A 0.1 ml aliquot of the supernatant was incubated with 0.2 ml of test compound solution, 0.1 ml of ferrous sulfate (FeSO4 , 10 ␮M) and 0.1 ml of ascorbic acid (0.1 mM) at 37 ◦ C for 1 h.

W. Ghali et al. / Industrial Crops and Products 44 (2013) 111–118

After adding 0.5 ml of trichloroacetic acid (TCA 28%, w/v) and 0.38 ml of thiobarbituric acid (TBA, 2%, w/v) the mixture was heated at 80 ◦ C for 20 min to stop the reaction, then centrifuged at 3000 × g for 10 min to remove the precipitated proteins. The color intensity of the (MDA)–TBA complex in the supernatant was measured at 532 nm. Butylated hydroxytoluene (BHT; 0–2 mg/ml) served as positive control. Inhibition ratio = [(A0 − An )/A0 ] × 100; with A0 = absorbance of control and An = absorbance of sample. 2.9. Protein carbonylation (ex vivo assay) The protein oxidation was carried out using fresh brain homogenate. The formation of carbonyl groups due to oxidative stress caused in vitro by the Fenton’s reaction, in the absence or presence of extracts, was assessed by a standard DNPH-coupled spectrophotometric method (Mishra et al., 2004). Different concentrations of test compound were added to aliquots of brain homogenate containing 1 mg of protein. Protein content of the homogenate was determined by Bradford method. Control was prepared by substituting extract with buffer. Samples were exposed to in vitro Fenton reactant (Fe2+ , H2 O2 ) generated free radicals. After that, the proteins were precipitated with ice chilled 10% TCA. The pellet was suspended in 0.2% 2,2 -dinitrophenyl hydrazine (DNPH) in 2 N HCl and incubated at room temperature for 2 h. Proteins were re-precipitated with TCA, excess DNPH was removed with several washes of 50% ethyl acetate in ethanol, the protein pellet was dissolved in 6N guanidine hydrochloride and the absorbance was measured at 370 nm (A). The results were expressed in terms of inhibition of formation of carbonyls/mg of protein. 2.10. DNA nicking assay A DNA nicking assay was assessed using supercoiled pUC19 plasmid DNA prepared from Top10 bacteria using extraction kit (Promega, Madison, WI). Plasmid DNA (0.2 ␮g) was added to Fenton’s reagents (30 mM H2 O2 , 50 ␮M ascorbic acid, and 80 ␮M FeCl3 ) containing different concentrations of hydro-alcoholic extract and the final volume of the mixture was brought up to 20 ␮l. The reaction mixture was then incubated for 30 min at 37 ◦ C and agarose gel electrophoresis was employed to follow Puc19 DNA damage. Agarose gel (1%) was prepared in 130 mM Tris–borate/2.5 mM EDTA (TBE) buffer. Ethidium bromide was included in the gel preparation to unable the visualization of the DNA bands in a UV transilluminator. The gel was submerged in an electrophoresis tank filled with TBE buffer. Control and samples were mixed with loading dye (0.25% bromophenol blue and 30% glycerol) and loaded into the wells. Electrophoresis was carried out at 75 V for 1 h 30–2 h to separate the different form of DNA plasmid. Also, effect of Jatropha extract on induction of strand breaks in plasmid DNA in the presence of copper sulfate was studied following the method of Thibodeau et al. (2001). Briefly a reaction mixture (20 ␮l) containing 200 ng of pUC19 DNA, 100 ␮M copper sulfate and varied concentrations of extract, was incubated at 37 ◦ C for 1 h. DNA samples were analyzed by electrophoresis under the same conditions previously mentioned.

113

for 1 h at 37 ◦ C. Thereafter, 2 volumes of solution containing 15% TCA and 1% thiobarbituric acid (TBA) were added, incubated in boiling water bath for 20 min, cooled and the absorbance of the resulting pink colored chromogen was measured at 532 nm. 2.12. Cells culture Experiments were performed using two tumor cells lines A549 (human lung adenocarcinomic cells), PC12 (rat pheochromocytoma),obtained from the American Type Culture Collection (Rockville, MD), and one kind of normal cells, cerebellar granule cells obtained from primary cells culture from 8 days post-natal rat. A549 cancer cells were maintained in RPMI medium supplemented with 10% heat-inactivated FBS, 100 units/ml penicillin and 100 mg/ml streptomycin. PC12 cells were grown in RPMI 1640 containing 10% heat-inactivated horse serum, 5% heat-inactivated fetal bovine serum and 1% penicillin/streptomycin antibiotic mixture. Cerebellum granule cells were dissociated from pooled 8-day-old rat cerebella, plated on coated plate with poly-d-lysine (5 tug/ml), and grown in basal modified Eagle’s medium containing 10% heatinactivated fetal calf serum, gentamycin (100 tug/ml), and 25 mM KCl. All cultured cells were incubated at 37 ◦ C in a humidified incubator with an atmosphere of 95% air and 5% carbon dioxide. Cells were subcultured two or three times a week. 2.13. Cytotoxicity and cell viability analysis (MTT assay) The MTT assay (a tetrazolium salt, reduction assay) was performed to evaluate cell viability (Mosmann, 1983). The cells seeded at 1 × 105 cells−1 ml−1 in 96-well tissue culture plates were allowed to attach and recover for at least 24 h varying concentrations of Jatropha extract (12.5–150 ␮g/ml) were added to each well. A negative control containing culture medium alone was also evaluated. After 24 h of treatment, the plates were incubated with the MTT solution (final concentration 0.5 mg/ml) for 3 h. After the medium had been discarded, 100 ␮l of DMSO was then applied to the well to dissolve the dark-blue formazan crystals in intact cells. Absorbance at 570 nm was measured on a Flextation3 reader. Cell viability was expressed as a percentage of untreated cells. The results were expressed as a percentage of the negative control which was designated as 100%. All assays were performed using samples in triplicate and repeated three times. 2.14. Statistical analysis The results presented are expressed as the mean ± standard deviation of three independent experiments (n = 3). The data were submitted to multiple variance analysis (ANOVA) and the post hoc Tukey test, using the GraphPad Prism 2.01 software program. A value of p < 0.05 was considered statistically significant for all the parameters evaluated. 3. Results and discussion

2.11. Estimation of hydroxyl radicals generation by jatropha extract

3.1. Total phenolic and flavonoid contents

Generation of hydroxyl radicals by Jatropha extract in the presence of 100 ␮M of copper sulfate was studied in vitro using 2-deoxyribose degradation assay (Klein et al., 1991). To 1 ml of reaction volume containing 5 mM 2-deoxyribose and 100 ␮M copper sulfate, varied concentrations of extract were added and incubated

The phenolic content in the selected plant extracts, evaluated using Folin–Ciocalteu method, is presented in Table 1. The content of total phenols in extracts, expressed as gallic acid equivalents (GAE), varied between 133.09 ± 1.4 and 311.57 ± 3.47 mg/g of dry extract.

114

W. Ghali et al. / Industrial Crops and Products 44 (2013) 111–118

Table 1 The total phenolics and flavonoids content of examined Jatropha podagrica’s organs. Plant organs

Extract

Leavesa Seedsa

MeOH/H2 O (80:20)

a b

Total phenolic content mg GAEb /g dry extract

Flavonoid content mg catechin/g dry extract

133.09 ± 1.4 311.57 ± 3.47

77.53 ± 10.75 172.4 ± 5.09

Average and standard deviation of triplicate analysis. GAE – gallic acid equivalent.

The flavonoid content (expressed as catechin equivalents: mg Catechin/g of dry extract) is given in Table 1. The greatest quantity of flavonoid compounds was found in Jatropha’s seeds.

defined as the concentration of trolox having equivalent antioxidant activity and expressed as mM/g of dry matter (Fig. 2).

3.2. DPPH radical scavenging activity 3.3. Reducing power The ability of hydro-alcoholic Jatropha extracts to scavenge the DPPH radicals was tested. The DPPH radical has been widely used to test the ability of compounds as free-radical scavengers or hydrogen donors and to evaluate the antioxidative activity of plants extracts and foods (Porto et al., 2000). As shown in Fig. 1, the extract significantly inhibited the activity of DPPH radicals in a dose-dependent manner. Our results demonstrated that Jatropha leaf extract has higher free radical scavenging activity than Jatropha seeds extract. The maximal inhibition of the anti-DPPH radical activity was calculated at 420 ± 0.077 ␮g/ml of Jatropha leaf extract; however, Jatropha seeds extract showed its maximal inhibition activity with120 ␮g/ml and then the activity remained constant. The concentration of each extract required to achieve 50% of DPPH radical reduction (IC50 ) was 78.19 ± 0.22 ␮g/ml and 71.34 ± 0.29 ␮g/ml respectively for leaf and seeds extracts. The antioxidant activity of extracts was also estimated by the trolox equivalent antioxidant capacity assay. The unit of TEAC is

The reducing power of a compound generally depends on the presence of molecules which showed an antioxidative potential by donating a hydrogen atom to break the free radical chain. The antioxidants present in J. podagrica leaves and seeds extracts reduced the Fe3+ /ferricyanide complex to the ferrous form. Fig. 3 shows the reductive capacity of the J. podagrica extracts compared to ascorbic acid. The increment in reducing power of the hydroalcoholic extracts should be due to the increase in the concentration of hydrogen donors. The activity of ascorbic acid was lower than the tested seeds extract. This is in line with other workers who had similar results (El Diwani et al., 2009). The leaves extracts of Jatropha showed a moderate reducing property (Fig. 3). This statement, in addition to DPPH assay results, could indicate that the leaves extract probably contains more electron donors reacting with free radicals. However, the seeds extract may chelate metal ions rather than scavenging hydroxyl radicals directly. The reducing power of organs correlated well with the polyphenol content (r seeds = 311.57 mg/g and r leaves = 133.09 mg/g; the results are expressed in mg of gallic acid equivalent/g dry matter).

3.4. Superoxide anions scavenging activity

Fig. 1. Free radical scavenging activity of Jatropha leaf and seeds extracts as determined by DPPH method. The reported values are means of triplicate determinations (n = 3) ± standard deviation.

The results from superoxide anion scavenging activity assay showed that J. podagrica extracts inhibited Nitro Blue Tetrazolium (NBT) reduction efficiently. In fact, leaf extract inhibited the production of superoxide anions by 50% when 1.29 ␮g/ml of the leaves extract, or 1.48 ␮g/ml of seed extract, was added to the reaction solution (Table 2). However J. podagrica extract alone did not change the absorbance of the reaction mixture containing only NBT, suggesting that the extract did not directly reduce the NBT.

Fig. 2. Sample content in trolox equivalent (mM trolox/g dry matter).

W. Ghali et al. / Industrial Crops and Products 44 (2013) 111–118

115

Fig. 3. Reducing power for Jatropha podagrica organs extracts and standard (ascorbic acid). Results are means of triplicate determinations (n = 3) ± standard deviation.

3.5. DNA nicking assay Hydroxyl radical generated by the Fenton’s reaction are also known to cause induced oxidative breaking in DNA stands to yield to its open circular or relaxed forms. To further measure the scavenging effect of J. podagrica extract on Fe3+ -dependent hydroxyl radicals; we investigated whether the extract could reduce Fe3+ -dependent DNA nicking (Fig. 4(A)). When pUC19 plasmid DNA was added to the reaction mixture, an increase in the formation of single-stranded nicked DNA (Form II) and of double-stranded nicked and linear DNA (Form III) was observed. However, the addition of only 20 ␮g of Jatropha extract to the reaction mixture helped keeping DNA into his supercoiled form (Form I). Consequently, the treatment leaded to disappear Fe3+ -mediated Form III DNA formation and reduce Form II DNA formation (Fig. 4(A), lanes 4–8). These results indicate that the Jatropha hydro-alcoholic extract effectively reduced the oxidative stress on susceptible biomolecules, such as DNA. Also, the addition of only J. podagrica extract in a relatively high dose (1 mg) to the plasmid DNA solution, did not induce any visual damage by converting of fast migrating supercoiled plasmid DNA to slow migrating relaxed form.

3.6. Plasmid damage and metal ions In the presence of 100 ␮M copper sulfate single and double strand breaks in plasmid DNA were induced, by the formation of open circular and linear forms (Fig. 4(B), lane 2). However, addition of different concentrations (50–200 ␮g/ml) of J. podagrica hydro-alcoholic extract to plasmid DNA in the presence of 100 ␮M of copper sulfate and under the buffer condition used in the present study did not result in significant increase in strand breaks

(Fig. 4(B), lanes 3–5). Similarly pUC19 DNA incubated with Jatropha extract (1000 ␮g/ml) also did not induce any significant nicks in DNA (Fig. 4(B), lane 1). This test was used to establish if polyphenolic compounds could generate DNA damage or other cytotoxic effects in specific conditions. Generally, properties of polyphenolics result from their ability to auto-oxidize, particularly in the presence of free metal ions, resulting in reduction of reactive hydroxyl radicals (Burkitt and Duncan, 2000). Also, Shukla et al. (2006) found that their extract REC-2001 in the presence of copper sulfate generates hydroxyl radicals and causes strand breaks in plasmid DNA in a dose dependant fashion. However, Jatropha hydro-alcoholic extract did not enhance the damages caused by the free copper sulfate present in the plasmid DNA solution.

3.7. Hydroxyl radicals generation The 2-deoxyribose degradation assay was used to estimate the hydroxyl radical generation potential of studied extract. Results revealed that Jatropha extract moderately generated hydroxyl radicals in the presence of 100 ␮M copper sulfate. Until the concentration 200 ␮g/ml, Jatropha extract did not exerted significant pro-oxidant effects, when added to the reaction solution, by generating free hydroxyl radicals compared to initial state generated by the addition of copper sulfate (Fig. 4(C)).

3.8. Inhibition of Lipid peroxidation and protein carbonylation assays Lipid peroxidation is a reaction between the hydroxyl radicals and unsaturated fatty acid side chains of lipids and phospholipids (Vermerris and Nicholson, 2006), catalyzed by transition-metal

Table 2 Antioxidant activity of Jatropha podagrica’s extracts. Plant organs

IC50 (␮g/ml) a DPPH scavenging activityb

Superoxide anion scavenging activityb

Leaves

78.19 ± 0.22

1.29 ± 0.133

Seeds

71.34 ± 0.29

1.48 ± 0.071

a b c

Average and standard deviation of triplicate analysis. % Inhibitory activity. Inhibition activity of total plant extract on lipid and protein oxidation.

Lipid peroxidationc

Protein oxidationc

98.55 ± 0.23

251.38 ± 0.038

116

W. Ghali et al. / Industrial Crops and Products 44 (2013) 111–118

Fig. 4. (A) Effect of total Jatropha hydro-alcoholic extract on DNA nicking caused by hydroxyl radical. The DNA nicking reaction was initiated by adding 0.2 ␮g of PUC plasmid DNA to Fenton’s reaction solution in the absence (lane 2) or presence (lanes 3–9) of extract (JHA). Lane 1: 0.2 ␮g pUC19 plasmid DNA, lane 2: pUC19 plasmid + Fenton’s reactive, lane 3: pUC19 + 25 ␮g/ml JHA extract + Fenton’s reactive, lane 4: pUC19 + 50 ␮g/ml JHA extract + Fenton’s reactive, lane 5: pUC19 + 100 ␮g/ml JHA extract + Fenton’s reactive, lane 6: pUC19 + 150 ␮g/ml JHA extract + Fenton’s reactive, lane 7: pUC19 + 200 ␮g/ml JHA extract + Fenton’s reactive, lane 8: pUC19 + 1000 ␮g/ml JHA extract + Fenton’s reactive, lane 9: pUC19 + 1000 ␮g/ml JHA extract. (B) Effect of different concentrations of Jatropha hydro-alcoholic extract (JHA) on induction of strand breaks on plasmid DNA in the presence of copper sulfate. Lane 1: 0.2 ␮g pUC19 DNA, lane 2: pUC19 DNA + 100 ␮M copper sulfate, lane 3: pUC19 DNA + 100 ␮M copper sulfate + 50 ␮g/ml JHA extract, lane 4: pUC19 DNA + 100 ␮M copper sulfate + 100 ␮g/ml JHA extract, lane 5: pUC19 DNA + 100 ␮M copper sulfate + 200 ␮g/ml JHA extract. MM: molecular marker. (C): Effect of varied concentrations of Jatropha extract on generation of hydroxyl radicals and subsequent degradation of 2-deoxyribose measured as a number of folds increase in formation of thiobarbituric acid reactive substances (TBARS). All values are mean ± SD of three parallel observations.

ions, and is considered to be the main cause of deterioration of food quality (Raza et al., 2009). Chemical oxidation induced by the Fenton reaction (Fe2+ /H2 O2 ) increased lipid oxidation. The inhibition of this oxidation by the extract was monitored by TBA-RS formation in brain homogenate solution in absence and presence of different concentrations of extract. The IC50 values of lipid peroxidation inhibition measured are reported in Table 2. Also, a minimal concentration of Jatropha extract (50 ␮g/ml) showed a high protective potential through lipids with 2838 ± 0.20% inhibition of peroxidation. Brain homogenate was also subjected to Fenton’s reactive in absence or presence of Jatropha extract to assess protein carbonylation. Under the effect of oxidative stress reactive oxygen species can induce the cleavage of peptide bonds of proteins. The hydroxyl radical generated by H2 O2 , reacts with proteins to form water and an alkyl radical which can then bind to other alkyl radicals to form aggregates of proteins or reacted with oxygen to generate peroxyl radical alkyl. Alkyl peroxide can then react with Fe (II) to produce an alkoxyl radical. Inhibition of protein oxidation is probably due to the antioxidant activity of the studied extract which secures the carboxyl groups and peptide bonds. The results indicated that in the presence of Jatropha extract, even in low doses, the protein carbonylation decreased effectively reaching 81, 64 ± 1, 03% of inhibition. From these results the IC50 values were estimated and listed in Table 2.

The results indicated that Jatropha extract exhibit a good protection toward cellular proteins and lipids against ROS damages. Results also showed that this high protection was detected at a low concentration of the total extract as shown by the IC50 values. Both protein carbonylation and lipid oxidation proceeds though free radical mechanism, therefore it is not surprising that extracts that scavenge specific free radicals are effective inhibitors of protein carbonylation and lipid oxidation. Those properties can be exploited in several scales including food and pharmaceutical industry

3.9. Antiproliferative effect of Jatropha extract on tumor cells The antiproliferative effect of J. podagrica extract (JE) was determined on two cancer cell lines (PC12 and A549). Cells were treated at different concentrations of studied extract (0–100 ␮g/ml) for 24 h. Cells incubated with 0.2% DMSO was used as a control. The two lines presented a high susceptibility to Jatropha extract. Treatment concentrations greater than 12 ␮g/ml presented a dose-dependent reduction in cell viability greater than 20%. At the highest concentration evaluated, the reduction in cell viability was greater than 80%. Jatropha extract was cytotoxic even at low concentration and the cell viability response was concentration dependent (Fig. 5(A)).

W. Ghali et al. / Industrial Crops and Products 44 (2013) 111–118

117

Fig. 5. (A) Effects of Jatropha extract on PC12 and A549 cell viability. Cytotoxicity was determined using MTT assay after 24 h treatment with the indicated concentrations. Values are expressed by mean ± S.D. (n = 3) 5. (B) Cytoprotective effect of Jatropha extract on neuronal cells. Cell viability was determined using MTT assay after 3 h, 12 h and 24 h of treatment with indicated concentrations. Values are expressed in mean ± SD (n = 3).

3.10. Cytotoxicity on cerebellar granule cells For in vitro evaluation of cytotoxicity of JE on normal granule cells, the use of primary cells cultures is very common, including granulated cerebellum used in this experiment. When treated with different concentrations of JE (0–150 ␮g/ml), viability of granulated neurons still normal compared to non treated cells. And after an exposure of these cells to JE for 3 h, 12 h and 24 h, no significant diminution on cells viability or number was observed (Fig. 5(B)). It indicates that Jatropha’s extract is not toxic for normal cells, despite its antiproliferative effect on tumor cells. This extract could be used for several tests to evaluate its possible antigenotoxicity/antimutagenicity effects on cells treated with oxidative stress factors and to study this propriety in an in vivo model. 4. Conclusion The results of this study indicate that the hydro-alcoholic extracts of J. podagrica possessed a high phenolic content and antioxidant activity and a remarkable protective effect on DNA, proteins and lipids against severe damages caused to cells by oxidative stress, leading to proteins degradation, lipids oxidation, or DNA nicking. Those damages could engender cells death and

mutagenesis. The studied extract showed also protective properties on whole functional cells, and did not show a cytotoxic effect on granulated neurons at relatively high concentration. When treated with JE, proliferation of tumor cell lines PC12 and A549 stopped and cell viability decrease to reach 20% in 24 h of treatment. In absence of Jatropha extract, the studied tumoral cells continue to grow and multiply. In conclusion, J. podagrica extract showed good anti-proliferative properties against studied cancer cells lines and in parallel it exhibits no cytotoxicity on normal cells. These in vitro effects reported here must be more investigated to elucidate the molecular mechanism responsible for anticancer/protective proprieties of JE and it is valuable for further investigation including elucidation of active components. Acknowledgment This work was supported by the Tunisian Minister of High Education and Scientific Research. References Aiyelaagbe, O.O., Adesogan, E.K., Ekundayo, O., Hassanali, A., 1998. Anti-feedant activity of Jatropha podagrica roots. Fitoterapia 69, 175–176. Beauchamp, C., Fridovich, I., 1971. Superoxide dismutase: improved assays and an assay applicable to acrylamide gels. Anal. Biochem. 44, 276–287.

118

W. Ghali et al. / Industrial Crops and Products 44 (2013) 111–118

Burkill, H.M., 1994. The useful plants of west tropical Africa. Roy. Bot. Gard. 2, 90–94. Burkitt, M.J., Duncan, J., 2000. Effects of trans-resveratol on copper dependent hydroxyl radical formation and DNA damage; evidences for hydroxyl radical scavenging and a novel glutathione sparing mechanism of action. Arch. Biochem. Biophys. 381, 253–263. Cai, Y.Z., Sun, M., Corke, H., 2003. Antioxidant activity of betalains from plants of the Amaranthaceae. J. Agric. Food Chem. 51, 2288–2294. Dewanto, V., Wu, X., Adom, K.K., Liu, R.H., 2002. Thermal processing enhances the nutritional value of tomatoes by increasing total antioxidant activity. J. Agric. Food Chem. 50, 3010–3014. El Diwani, G., El Rafie, S., Hawash, S., 2009. Antioxidant activity of extracts obtained from residues of nodes leaves stem and root of Egyptian Jatropha curcas. Afr. J. Pharm. Pharmacol. 3 (11), 521–530. Esterbauer, H., 1996. Estimation of peroxidative damage: a critical review. Pathol. Biol. 44, 25–28. Fattouch, S., Caboni, P., Coroneo, V., Tuberosos, C., Angioni, A., Dessi, S., Marzouki, M.N., Cabras, P., 2008. Comparative analysis of polyphenolic profiles and antioxidant and antimicrobial activities of Tunisian pome fruit pulp and peel aqueous acetone extracts. J. Agric. Food Chem. 56, 1084–1090. Fattouch, S., Fattouch, F.R., Gil Ponce, J.V., Forment, J.V., Lukovic, D., Marzouki, M.N., Vidal, D.R., 2010. Concentration dependent effects of commonly used pesticides on activation versus inhibition of the quince (Cydonia oblonga) polyphenol oxidase. Food Chem. Toxicol. 48, 957–963. García-Pérez, M.E., Royer, M., Duque-Fernandez, A., Diouf, P.N., Stevanovic, T., Pouliot, R., 2010. Antioxidant, toxicological and antiproliferative properties of Canadian polyphenolic extracts on normal and psoriatic keratinocytes. J. Ethnopharmacol. 132 (1), 251–259. Girotti, A.W., 1994. Photodynamic lipid peroxidation in biological systems. Photochem. Photobiol. 51, 497–509. Gyamfi, M.A., Yonamine, M., Aniya, Y., 1999. Free-radical scavenging action of medicinal herbs from Ghana: Thonningia sanguine on experimentally-induced liver injuries. Gen. Pharmacol. 32, 661–667. Halliwell, B., 1994. Free radicals, antioxidants, and human disease: curiosity, cause, or consequence? Lancet 344, 721–724. Halliwell, B., Gutteridge, J.M.C., 1999. Free Radicals in Biology and Medicine. Oxford Press, Oxford.

Kitts, D.D., Wijewickreme, A.N., Hu, C., 2000. Antioxidant properties of a North American ginseng extract. Mol. Cell. Biochem. 203, 1–10. Klein, S.M., Cohen, G., Cederbaum, A.I., 1991. Production of formaldehyde during metabolism of dimethyl sulphoxide by hydroxyl radical generating system. Biochemistry 20, 6006–6012. Mishra, H.S., Khairnar, N.P., Barik, A., Priyadarsini, K.I., Mohan, H., Apte, S.K., 2004. Pyrroloquinoline and quinone a reactive oxygen species scavenger in bacteria. FEBS Lett. 58, 26–30. Mosmann, T., 1983. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods 65, 55–63. Ohkawa, H., Ohishi, N., Yagi, K., 1979. Assay for lipid peroxides in animal tissues by thio-barbituric acid reaction. Anal. Biochem. 95, 357–358. Porto, C.D., Calligaris, S., Celloti, E., Nicoli, M.C., 2000. Antiradical properties of commercial cognacs assessed by the DPPH test. J. Agric. Food Chem. 48, 4241–4245. Raza, S.A., Rashid, A., William, J., Najaf, S., Arshad, M., 2009. Effect of synthetic antioxidant on shelf life of locally manufactured butter known as Makhan in Pakistan. Bih. Biol. 3 (2), 91. Shukla, S.K., Choudhary, P., Kumar, I.P., Afrin, F., Puri, S.C., Qazi, G.N., Sharma, R.K., 2006. Radiomodifying effects of a fractionated extract of Podophyllum hexandrum: a mechanistic study. Environ. Toxicol. Pharmacol. 22, 113–120. Shyamala, B.N., Sheetal, G., Jyothi, L.A., Jamuna, P., 2004. Leafy vegetable extracts antioxidant activity and effect on storage stability of heated oils. Innovat. Food Sci. Emerg. Technol. 6, 239–245. Sies, H., 1997. Antioxidants in Disease, Mechanisms and Therapy. Academic Press, New York. Thibodeau, P.A., Kocsis-Bederd, S., Courteau, J., Niyonsenga, T., Paquette, B., 2001. Thiols can either enhance or suppress DNA damage induction by catecholestrogens. Free Radical Biol. Med. 30, 62–73. Vermerris, W., Nicholson, R., 2006. Phenolic Compound Biochemistry. Springer, Dordrecht. Wolfe, K., Wu, X., Liu, R.H., 2003. Antioxidant activity of apple peels. J. Agric. Food Chem. 51, 609–614. Zheng, W., Wang, S.Y., 2001. Antioxidant activity and phenolic compounds in selected herbs. J. Agric. Food Chem. 49, 5165–5170.